Recurrent microdeletion syndromes (rMDS), such as those associated with 16p11.2, 1q21 and 22q11.2, represent a major component (10-15%) of the genetic etiology of neurodevelopmental disorders, including autism spectrum disorder (ASD). In each rMDS, a distinct set of genes is reproducibly reduced to haploid dosage due to non-allelic homologous recombination (NHAR) mediated by flanking segmental duplications. The neurodevelopmental phenotypes in rMDS could theoretically derive primarily from a single gene driver, as we have shown is often the case for non-recurrent MDS, or from the combined effects of multiple genes that are dysregulated in concert. This project will test the hypothesis that the transcriptional effects of these three common rMDS converge on a small number of pathways/processes that are critical for abnormal neurodevelopment. It will further evaluate whether there are individual genetic drivers responsible for the dysregulated networks associated with these rMDS, and whether the transcriptional changes that occur can be rescued either by directly re-introducing the driver gene into a cellular system or through trans-rescue of secondary networks. The project will capitalize on our recent advances in CRISPR/Cas9 genome editing to efficiently generate large deletions in induced pluripotent stem cells (iPSC) using a novel dual-guide approach. These isogenic cells eliminate the confound of differing genetic backgrounds and thereby provide a powerful assay system. Specifically, we aim to compare the transcriptional consequences of full deletion in these three rMDS regions and identify overlapping genes/pathways that are altered due to haploinsufficiency for each full microdeletion (Aim 1). We will then seek to identify key genetic drivers within each rMDS by systematic single gene ablation within the rMDS region (Aim 2). Finally, we will test whether critical network alterations can be rescued by re-introducing a single driver gene or manipulating hub genes within dysregulated networks (Aim 3). At its conclusion, this study will have deconstructed three of the most common rMDS to yield critical neurodevelopment-associated networks that can be rescued by secondary manipulation, thereby offering a direct route to the discovery of neuronal biomarkers and targeted therapeutic intervention.